JPH026761B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a polysaccharide and a method for producing the same, and more specifically, a polysaccharide derived from Fukurotake, which is a basidiomycete, and the extraction and collection of this polysaccharide from Fukurotake. It is related to its manufacturing method. [Problems to be solved by conventional techniques and inventions] Living organisms that can reproduce and repair themselves use many kinds of low-molecular substances such as amino acids and lipids as well as macromolecules such as proteins, nucleic acids, and polysaccharides in order to maintain their functions. It is made up of matter. Polysaccharides are therefore widely occurring in nature. It is widely used as food, medicine, etc. Among these polysaccharides, polysaccharides derived from basidiomycetes have recently attracted attention as anticholesterol drugs, immunostimulants, and the like. As such polysaccharides, lentinan from Shiitake mushroom and schizophyllan from Swahirotake mushroom are particularly well known. All of these are said to have β-1,3-glucan as the main chain, with glucose branched through α-1,6-bonds. It is said that basidiomycetes, pachyman from Sclerotium, and scleroglucans from microorganisms of the genus Sclerotium have similar structures. In addition, the polysaccharide produced by microorganisms belonging to the genus Pestalotia is β-1,3
-D-glucopyranosyl group and D-glucopyranosyl-D- with β-1,6-bonds in the main chain of glucan
It is said to have a structure with side chains of glucopyranosyl groups, but the number of side chains is extremely large, and the main chain
It is said to be about 55 to about 75 pieces per 100 pieces (Japanese Unexamined Patent Publication No. 34702/1983). It is known that anti-tumor polysaccharides can be obtained from Basidiomycetes, Cysophyllus spp., and Pseudophytes belonging to the genus Fukurotake by extraction with solvents such as water, lower alcohols, lower ketones, or ethers (Japanese Patent Application Laid-Open No. 1983-1997) - Publication No. 38723). However, its structure
Its physical properties are completely unknown. It is also known that a hypotensive substance called Santanin 2 can be prepared by extracting the mycelium of Fukurotake with a dilute alkaline solution (Japanese Unexamined Patent Publication No. 47483/1983). However, this substance is not composed only of polysaccharides. [Means for solving the problem] The present invention essentially consists of a first repeating unit represented by the formula [→3)β-D-Glu(1]→... () The second repeating unit and expression expressed by It consists of a third repeating unit represented by
and the number of repeating units of the third (formula ()) is about 4 to about 12 on average per 100 repeating units of the second (formula ()) and the third
The sum of the repeating units in (formula ()) is also approximately
16 to about 33 polysaccharides. [Function] The physical and scientific properties of the polysaccharide of the present invention are as follows. (1) Solubility Soluble in aqueous alkaline solutions and dimethyl sulfoxide, and substantially insoluble in methanol, ethanol, n-butanol, acetone, benzene, toluene, ethyl acetate, propylene glycol, pyridine, etc. Although the dried polysaccharide of the present invention is sparingly soluble in water, it can be made solubilized in water by applying operations such as ultrasonication. It is also slightly soluble in aqueous salt solutions. When it is dissolved in an alkaline aqueous solution at a low concentration and then neutralized, it remains dissolved. (2) Optical rotation The specific optical rotation of the polysaccharide of the present invention in a 0.5N aqueous sodium hydroxide solution (0.5%) is [α] ^D ₂₅ :about -12°. (3) Elemental analysis values If carefully and thoroughly dried, it will give a value close to the value calculated from CnH ₂ nOn, but it usually contains a small amount of water, C: 43-45% H: 5.6-6.4% N: Limit of quantification Give the following value. Halogens and sulfur are not detected. (4) Infrared absorption analysis Figure 1 shows the infrared absorption spectrum obtained by the KBr tablet method. The absorption at 896 cm ^-1 is characteristic of the β-bond of D-glucose residues. (5) C ¹³ −NMR analysis δ value: 103.6, 86.8, 76.9, 75.2, 74.2,
(ppm) Shows peaks at 73.3, 70.7, 69.0 and 61.4. (6) Color reaction Anthrone reaction: Positive Ninhydrin reaction: Negative Dish carbazole reaction: Negative (7) Molecular weight In gel permeable high performance liquid chromatography using Tris hydrochloride buffer with a concentration of 0.1 mol/PH8.6 as the mobile phase. It mainly elutes with a molecular weight of about 300,000 to about 2.8 million. (8) Reaction with cetylpyridinium chloride Does not form a precipitate with cetylpyridinium chloride in aqueous solution. (9) Constituent Saccharide The polysaccharide of the present invention was dissolved in a 1N sulfuric acid aqueous solution at 100%
After hydrolysis at °C, only glucose is detected by paper chromatography and, after derivation into alditol acetate, by gas chromatography. (10) Bond structure According to methylation analysis, the molar ratio is 2,3,4,6-tetra-O-methylglucose 1 2,4,6-tri-O-methylglucose about 2.7 to about 5.0 2,3, 4-tri-O-methylglucose from about 0.10 to about 0.35 and 2,4-di-O-methylglucose from about 0.7 to about 1.3. Enzymatic decomposition using exo-type β-1,3-glucanase produces glucose and gentiobiose. In addition, it is sufficiently oxidized with sodium metaperiodate,
After reduction with sodium borohydride, mild conditions,
For example, 0.1 to 0.2N sulfuric acid, 80 to 100℃,
When hydrolyzed for about 1 to 2 hours, linear β-1,3-glucan from which glucose groups corresponding to side chains have been removed is obtained. This one was found to be 2 in methylation analysis.
Gives 4,6-tri-O-methylglucose and trace amounts of 2,3,4,6-tetra-O-methylglucose. When this linear β-1,3-glucan is enzymatically decomposed with exo-type β-1,3-glucanase, only glucose is produced. Therefore, the main chain of the polysaccharide of the present invention is substantially β-1,
Consists only of 3-linked glucose residues. From this, the polysaccharide of the present invention has continuous β-
Consists of a main chain of 1,3-glucoside bonds, approximately β
One out of every three to six -1,3-bonded glucose residues has a side chain consisting mainly of a single glucose residue with a β-bond at the 6th position, but out of about three side chains, One is recognized to consist of two β-1,6-linked glucose residues. Therefore, it can be said that the basic structure of the polysaccharide of the present invention consists of repeating units of the above-mentioned general formula. Based on these facts, the polysaccharide of the present invention has a lower frequency of branching than the branched β-1,3-glucans obtained from known scleroglucans, schizophyllans, and wood ear fruiting bodies, and has a small amount of β during branching. ―
It is a polysaccharide with branches consisting of two or more consecutive glucose residues in 1,6-bonds. Furthermore, the frequency of branching is significantly lower than that of polysaccharides produced by microorganisms of the genus Pestalotia. Since the polysaccharide of the present invention cannot be extracted with water,
This is a different substance from the polysaccharide disclosed in Publication No. 57-38723. Similarly, the polysaccharide of the present invention is clearly a different substance from the aforementioned santanin 2, as the latter contains a large amount of nitrogen and is positive for the ninvitrin reaction (therefore, it is presumed to be a glycoprotein). It is. The polysaccharide of the present invention has immunostimulatory and antitumor effects, and can also be expected to have antiviral effects. The polysaccharide of the present invention can be easily prepared by extracting a basidiomycete belonging to the genus Volvariella, particularly Volvariella volcacea, with an aqueous alkaline solution. That is, the present invention also provides a method for producing the polysaccharide of the present invention by extracting Fukurotake with an aqueous alkaline solution. The Fukurotake used as a raw material in the method of the present invention may be either its fruiting body or mycelium.
These may be in a raw state or in a dried form. It is effective to shred raw materials before extraction to increase extraction efficiency. As the aqueous alkali solution used for extraction, for example, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide can be used. In particular, an aqueous sodium hydroxide solution is convenient. The concentration of the alkali used is not particularly limited, but it is preferably used within the range of about 0.1N to about 3N. The amount of alkaline aqueous solution used for extraction is usually about 5 to about 100 times the dry weight of the raw material per extraction. If this amount is too small, the extraction efficiency will be poor, and if it is too large, post-processing will be troublesome. In this extraction step, in order to prevent decomposition of the polysaccharide or denaturation such as peroxidation, it is preferable to perform the extraction under an atmosphere of an inert gas such as nitrogen. Furthermore, the reaction may be carried out in the presence of a reducing agent such as sodium borohydride. There are no particular limitations on the extraction temperature, but it is preferably about 30°C or lower. This is because polysaccharides having even more branches will be extracted if extracted at a higher temperature, for example, about 60 to 80°C, which is the temperature condition for normal hot alkaline extraction. The extraction operation may be repeated. The Fukurotake used in the present invention also contains many components for maintaining its own life within its bacterial body. Therefore, if the polysaccharide is directly extracted with an alkaline aqueous solution, various impurities will be included in the target polysaccharide. Although it is possible to purify the target polysaccharide from this extract, it requires complicated steps. Since the polysaccharide of the present invention cannot be extracted with a solvent such as water or alcohol, these impurities are removed using water, hot water, a buffered aqueous solution, alcohol, or a combination thereof prior to the step of extraction with an alkaline aqueous solution. It is preferable to keep it. In addition, this operation removes proteins that become contaminants during alkaline aqueous extraction.
Manganolactone, glycogen-like polysaccharide, etc. can be removed in advance. The alkaline aqueous solution extracted according to the method of the present invention is neutralized with an acid such as hydrochloric acid or acetic acid. Normally, in this state, the target polysaccharide is dissolved in the salt solution. Some precipitation may occur. If desired, a quaternary ammonium salt such as cetylpyridinium chloride may be added to this neutralized solution to precipitate contaminating substances with additivity as an insoluble precipitate. This precipitate is removed by conventional separation means such as filtration or centrifugation. The target polysaccharide can be obtained by dialyzing the thus obtained neutralized solution as it is or after concentration against water and drying the dialysate. Add alcohol to the neutralized solution as it is or after concentrating it.
A precipitant such as acetone may be added, and the resulting precipitate may be dialyzed against water, if desired, and dried to obtain the desired polysaccharide as a solid. As a drying method at that time, suitable drying means such as reduced pressure drying, freeze drying, and fog drying can be used. [Example] The present invention will be explained in more detail below with reference to Examples. Example 1 100 g of air-dried fruiting bodies of Fukurotake were soaked overnight in 0.1M phosphate buffer 1 of pH 7.0 containing 0.9% sodium chloride, and then crushed using a mixer and a homogenizer. Further, the same phosphate buffer 2 was added to this, followed by stirring and centrifugation for 4 hours. The obtained solid content was placed in the same phosphate buffer solution 3 and dispersed using a homogenizer, followed by stirring for 4 hours and centrifugation to obtain a precipitate. This precipitate was dispersed in 3 water and heated in an autoclave at 120°C for 30 minutes. After cooling, it was centrifuged to obtain a precipitate. This hot water extraction treatment was repeated four more times to almost completely extract and remove the water-soluble fraction. The water-insoluble fraction thus obtained was dispersed in a 1N aqueous sodium hydroxide solution 3 in which 5 g of sodium borohydride was dissolved. 25 under nitrogen flow
After stirring at °C for 4 hours, the mixture was centrifuged. This precipitate was subjected to repeated extraction operations using a 1N aqueous sodium hydroxide solution. Both sodium hydroxide extracts were combined and neutralized with concentrated hydrochloric acid to adjust the pH to 6.5. Cetylpyridinium chloride aqueous solution (10%) was added dropwise to the thus obtained extract while stirring until no further precipitate was formed.Then, centrifugation was performed at 10,000G for 30 minutes to remove the formed precipitate. did. An equal volume of methanol was added to this supernatant and stirred to precipitate the polysaccharide. This precipitate was washed with 1 methanol, added to 500 ml of distilled water, dispersed in a homomixer, and then dialyzed in running water for 5 days. The dialysis fluid was freeze-dried to obtain 13.0 g of the target product. Elemental analysis values C: 44.03% H: 6.05% N: Specific rotation below the limit of quantification [α] ²⁵ _D : -12° (concentration 0.5~, in 0.5N sodium hydroxide solution) Infrared absorption spectrum Figure 1 shows KBr This shows a Fourier transform infrared absorption spectrum obtained by the tablet method. The molecular weight was determined by gel permeation high performance liquid chromatography using a G5000PW column manufactured by Toyo Soda Kogyo Co., Ltd. using a Tris hydrochloride buffer with a molecular weight of 0.1M and a pH of 8.6 as the mobile phase.
It eluted at a retention time of 680,000. C ¹³ -NMR analysis, color reaction, and reaction with cetylpyridinium chloride The physical and chemical properties were the same as described above. Constituent sugars and bonding mode The obtained polysaccharide was methylated and further hydrolyzed. The obtained methylated sugar was converted into alditol acetate. This was analyzed by gas chromatography. The molar ratio of the obtained methylated sugar is 2,3,4,6-tetra-O-methylglucose
1 2,4,6-tri-O-methylglucose 3.76 2,3,4-tri-O-methylglucose 0.33 2,4-di-O-methylglucose 1.01 Separately, this polysaccharide was converted into exo-type β-1,3- Enzymatically digested with glucanase. Glucose and gentiobiose were obtained. The molar ratio was 1 mol of the former to 0.177 mol of the latter. Furthermore, this polysaccharide was sufficiently oxidized with sodium metaperiodate and reduced with sodium borohydride. This was further heated for 2 hours at 100℃ in sulfuric acid for 0.1 period.
After time hydrolysis, a linear glucan precipitate from which glucose groups corresponding to side chains had been removed was obtained. Methylation analysis of this precipitate was conducted in the same manner, yielding 2,4,6-tri-O-methylglucose and a trace amount of 2,3,4,6-tetra-O-methylglucose. Furthermore, when this precipitate was enzymatically decomposed with exo-type β-1,3-glucanase, only glucose was produced. Therefore, the obtained polysaccharide has the above-mentioned formulas (), ()
It consists of repeating units of and (), and the ratio of the number is per 100 of the total number of these repeating units,
There were 7 repeating units of formula (), and the total number of repeating units of formula () and repeating units of formula () was 21. Example 2 After carrying out the step before the alkaline aqueous solution extraction in the same manner as in Example 1, an alkaline aqueous solution extraction was carried out in the same manner as in Example 1 except that the extraction temperature was changed to 20°C. After adjusting the obtained extract to pH 6.5 with hydrochloric acid, a cetylpyridinium chloride aqueous solution (10%) was added in the same manner as in Example 1, and the resulting precipitate was removed by centrifugation. After dialyzing the supernatant in running water for 2 days, the dialyzed solution was concentrated under reduced pressure at 40°C to a concentration of 3. Add an equal volume of methanol to this and collect the resulting precipitate.
Centrifugation was performed at 10,000G for 30 minutes. 300ml of this sediment
of methanol and was stirred and centrifuged for about 1 hour to obtain a precipitate. After repeating this washing with methanol twice, the precipitate was dried under reduced pressure to obtain the desired product.
10.5g was obtained. The thus obtained polysaccharide was analyzed in the same manner as in Example 1. Elemental analysis values C: 44.31% H: 6.10% N: Molecular weight below the limit of quantification Eluted at a molecular weight of approximately 550,000. Methyl ratio sugar molar ratio 2,3,4,6-tetra-O-methylglucose 1 2.4,6-tri-O-methylglucose 4.26 2,3,4-tri-O-methylglucose 0.32 2,4-dimethylglucose -O-Methylglucose 1.01 Molar ratio of glucose and gentiobiose obtained by exo-type β-1,3-glucanase digestion 1:0.160 Ratio of repeating units 6 repeating units of formula () per 100 total units
, and the sum of the number of repeating units in equation () and the number of repeating units in equation () was 19. Other results were substantially the same as in Example 1. [Effect of the invention] Example of use of the polysaccharide of the present invention as an antitumor agent.
The effect on 180 types of tumors was tested. 5 x 16 ⁶ Sarcoma 180 ascites carcinoma cells were inoculated subcutaneously in the groin area per ICR mouse. The experimental group consisted of 6 animals. After the cancer cell transplantation, the drug was intraperitoneally administered in an amount of 0.1 ml once a day for 10 days from the next day.
For the test group, the polysaccharide of the present invention (obtained in Example 1) was dissolved in physiological saline at the dosage shown in Table 1, and for the control group, only physiological saline was administered. did. On the 35th day after tumor implantation, the cancer was excised and its weight was measured. The tumor suppression rate of each group was calculated using the following formula. Inhibition rate (%) = (1-average tumor weight of test group/average tumor weight of control group) ×100 The results are shown in Table 1. 【table】

[Brief explanation of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of the polysaccharide of the present invention.

Claims (1)

[Claims] 1. A first repeating unit essentially represented by the formula [→3)β-D-Glu(1]→ The second repeating unit and expression expressed by The third repeating unit represented by (in each formula, Glu
represents a glucopyranosyl group, and the number represents the bonding position), and the average number of third repeating units is about 4 to about 12 per 100 total of each of the first, second, and third repeating units. and a polysaccharide in which the sum of the numbers of the second repeating unit and the third repeating unit is about 16 to about 33. 2. The polysaccharide according to claim 1, which is a polysaccharide derived from Fukurotake. 3 Claims 1 or 2 which provide a molecular weight of about 300,000 to about 2,800,000 yen in gel permeation high performance liquid chromatography using a Tris hydrochloride buffer with a concentration of 0.1 mol/pH 8.6 as a mobile phase. polysaccharides. 4 Methylation analysis revealed that 2,3,4,6-tetra-O-methylglucose, 2,4,6-tri-
O-methylglucose, 2,3,4-tri-O-
The molar ratio of methylglucose and 2,4-di-O-methylglucose is 2,4,6 when 3,3,4,6-tetra-O-methylglucose is 1.
- Tri-O-methylglucose from about 2.7 to about
5.0, 2,3,4-tri-O-methylglucose in a ratio of about 0.1 to about 0.35 and 2,4-di-O-methylglucose in a ratio of about 0.7 to about 1.3. The polysaccharide described in any of the following. 5. Claims 1 to 4 which are soluble in aqueous alkaline solutions and dimethyl sulfoxide, and substantially insoluble in methanol, ethanol, n-butanol, acetone, propylene glycol, benzene, toluene, pyridine, and ethyl acetate. Polysaccharides listed. 6. The polysaccharide according to any one of claims 1 to 5, which is obtained by extracting Fukurotake with an aqueous alkaline solution. 7 Phthalotake is extracted with an alkaline aqueous solution to obtain the first repeating unit, which is essentially represented by the formula [→3) β-D-Glu(1]→ The second repeating unit and expression expressed by The third repeating unit represented by (in each formula, Glu
represents a glucopyranosyl group, and the number represents the bonding position), and the average number of third repeating units is about 4 to about 12 per 100 of the total number of first, second, and third repeating units. A method for producing a polysaccharide, which comprises collecting a polysaccharide in which the sum of the second and third repeating units is about 16 to about 33. 8. Claim 7, wherein the polysaccharide has a molecular weight of about 300,000 to about 2.8 million in gel permeation high performance liquid chromatography using a Tris-HCl buffer with a concentration of 0.1 mol/PH8.6 as the mobile phase. Manufacturing method described. 9 Polysaccharides were found to have 2,3,
4,6-tetra-O-methylglucose, 2,
4,6-tri-O-methylglucose, 2,3,
The molar ratio of 4-tri-O-methylglucose and 2,4-di-O-methylglucose is 2,4,6-tri-O-methylglucose, when 2,3,4,6-tetra-O-methylglucose is 1. -O-methylglucose approx.
2.7 to about 5.0, 2,3,4-tri-O-methylglucose about 0.1 to about 0.35 and 2,4-di-
9. The method of claim 7 or 8, wherein O-methylglucose is provided in a proportion of about 0.7 to about 1.3. 10 Claims 7 to 9, wherein the polysaccharide is soluble in aqueous alkaline solutions and dimethyl sulfoxide, and substantially insoluble in methanol, n-butanol, acetone, propylene glycol, benzene, toluene, and ethyl acetate. The manufacturing method according to any one of paragraphs. 11 Claims 7 to 10, wherein the aqueous alkali solution is an aqueous solution of an alkali metal hydroxide.
The manufacturing method according to any one of paragraphs. 12 Claims 7 to 1, wherein the concentration of the alkaline aqueous solution is about 0.1N to about 5N.
The manufacturing method according to any one of Item 1. 13. The production method according to any one of claims 7 to 12, wherein the extraction is carried out in the presence of a reducing agent. 14. The production method according to any one of claims 7 to 13, wherein the extraction is carried out at a temperature of about 30°C or less.